Project Summary Aedes aegypti is the principal mosquito vector of viruses that cause Zika, yellow fever, chikungunya, and dengue, the most widespread and significant arboviral disease in the world. Although mosquito control is the primary mechanism for disease control, insecticide resistance and a lack of support for mosquito control programs compromise existing strategies for managing Aedes mosquitoes. Females differ from males in morphological, physiological, and behavioral traits that are critical components of their ability to spread diseases. Researchers have therefore had a long-standing interest in the potential to manipulate genes that contribute to sexually dimorphic mosquito characters. Although genes that regulate sex-specification and development of mosquito sexual dimorphism may represent novel targets for vector control, many of these genes have not yet been functionally characterized in vector mosquitoes. Additionally, several new mosquito control technologies designed to eliminate large populations of mosquitoes are dependent upon efficient sex- sorting prior to mass release of male mosquitoes. However, affordable and effective methods of sex-sorting that can be employed for mass-rearing of insects at remote or resource-limited locations have unfortunately not yet been established. The proposed investigation addresses both the need for new sex-specific genetic targets, as well as the requirement for new sex-sorting techniques. A successful RNA interference pilot screen uncovered multiple female-specific larval lethal long non-coding RNA (lncRNA) genes. Although thousands of putative lncRNA genes have been identified in the A. aegypti genome, these genes, once considered dark matter, have not yet been functionally validated as lncRNA genes. This research program will invoke the use of newly developed yeast interfering RNA larvicide technology to functionally test the hypothesis that lncRNAs regulate A. aegypti sex-specific development. The proposed aims of this study include: (1) continued identification and characterization of lncRNAs that regulate sex-specific development, (2) generation of yeast interfering RNA larvicide strains corresponding to female-specific larval lethal lncRNA genes, and (3) down- selection and evaluation of female-specific yeast interfering RNA larvicides under simulated mass-rearing conditions. In addition to functionally validating lncRNAs as a new class of genes that regulate sex-specific development in mosquitoes, this study will promote the development of affordable, effective, and scalable female-specific yeast interfering RNA larvicide technology that could enhance the potential for mass-rearing male mosquitoes in remote and resource-limited regions throughout the world. It is anticipated that the proposed investigation will uncover many lncRNA genes that regulate sex-specific development and differentiation, and the full characterization of these genes will be pursued in future investigations.